Low saturated-fat sunflower and associated methods

ABSTRACT

Provided are sunflowers, parts thereof, cultures of, and seeds that are capable of producing sunflower oil that is low in saturated fat and, optionally, high in linoleic acid as well as associated methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.12/340,525, filed Dec. 19, 2008, pending, which claims the benefit ofthe filing date of U.S. application Ser. No. 61/015,591, filed Dec. 20,2007. This application is also related to U.S. application Ser. No.12/340,558, filed Dec. 19, 2008, pending.

FIELD OF THE INVENTION

The present invention relates to new and distinctive sunflowersproducing seeds that are low in saturated fat and, optionally, high inlinoleic acid as well as associated methods. The present inventionfurther relates to non-genetically modified, non-mutagenized sunflowershaving glyphosate resistance and associated methods.

BACKGROUND OF THE INVENTION

The cultivated sunflower (Helianthus annuus L.) is a major worldwidesource of vegetable oil. In the United States, approximately 4 millionacres are planted in sunflowers annually, primarily in the Dakotas andMinnesota.

The very rapid expansion over the last decade of acreage planted insunflower in the United States is due in part to several importantdevelopments in the field of sunflower breeding and varietalimprovement. One significant development was the discovery ofcytoplasmic male sterility and genes for fertility restoration, adiscovery that allowed the production of hybrid sunflowers. The hybridsthus produced were introduced during the early 1970s.

A description of cytoplasmic male sterility (CMS) and genetic fertilityrestoration in sunflowers is presented by Fick, “Breeding and Genetics,”in Sunflower Science and Technology 279-338 (J. F. Carter ed. 1978), thecontents of which are incorporated herein by reference.

Sunflower oil is comprised primarily of palmitic (16:0), stearic (18:0),oleic (18:1), linoleic (18:2) and linolenic (18:3) acids. While otherunusual fatty acids exist in plants, palmitic, stearic, oleic, linoleic,and linolenic acids comprise about 88% of the fatty acids present in theworld production of vegetable oils. (J. L. Harwood, Plant Acyl Lipids:Structure, Distribution and Analysis, 4 Lipids: Structure and Function,P. K. Stumpf and E. E. Conn ed. (1988).) Palmitic and stearic acids aresaturated fatty acids that have been demonstrated in certain studies tocontribute to an increase in the plasma cholesterol level, a factor incoronary heart disease. According to recent studies, vegetable oils highin unsaturated fatty acids, such as oleic and linoleic acids, may havethe ability to lower plasma cholesterol. Saturated fatty acids also havehigher melting points in general than unsaturated fatty acids of thesame carbon number, which contributes to cold tolerance problems infoodstuffs and can contribute to a waxy or greasy feel in the mouthduring ingestion. It is also known that food products made from fats andoils having less than about 3% saturated fatty acids will typicallycontain less than 0.5 gram saturated fat per serving and as a result canbe labeled as containing “zero saturated fat” under current labelingregulations. Thus, for a number of reasons, it is desirable to produce asunflower oil having low levels of palmitic and stearic acids and highlevels of oleic or linoleic acids.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, and better agronomic quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior because, for most traits, the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superiorsunflower cultivars and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same sunflower traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is due tothe breeder's selection, which occurs in unique environments, with nocontrol at the DNA level (using conventional breeding procedures), andwith millions of different possible genetic combinations beinggenerated. A breeder of ordinary skill in the art cannot predict thefinal resulting lines he develops, except possibly in a very gross andgeneral fashion. The same breeder cannot produce the same cultivar twiceby using the exact same original parents and the same selectiontechniques. This unpredictability results in the expenditure of largeamounts of research monies to develop superior new sunflower cultivars.

The development of new sunflower cultivars requires the development andselection of sunflower varieties, the crossing of these varieties, andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as pod color, flower color, pubescence color, or herbicideresistance which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, sunflower breeders commonly harvest seedsfrom each plant in a population and thresh them together to form a bulk.Part of the bulk is used to plant the next generation and part is put inreserve. The procedure has been referred to as modified single-seeddescent.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to remove seeds with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar can incur additional costs tothe seed producer, the grower, processor and consumer due to specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Sunflower, Helianthus annuus L., is an important and valuable fieldcrop. Thus, a continuing goal of plant breeders is to develop stable,high yielding sunflower cultivars that are agronomically sound. Acurrent goal is to maximize the amount of grain produced on the landused and to supply food for both animals and humans. To accomplish thisgoal, the sunflower breeder must select and develop sunflower plantsthat have traits that result in superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF SUMMARY OF THE INVENTION

The following embodiments are described in conjunction with systems,tools and methods which are meant to be exemplary and illustrative, andnot limiting in scope. In various embodiments, one or more of theabove-described problems have been reduced or eliminated, while otherembodiments are directed to other improvements.

According to the invention, there is provided a novel sunflower plantproducing seeds having low saturated fat content. This invention, inpart, relates to the seeds of sunflower having low saturated fatcontent, to the plants or plant parts, of sunflower plants producingseeds having low saturated fat content, and to methods for producing asunflower plant produced by crossing the sunflower plants producingseeds having low saturated fat content with itself or another sunflowercultivar, and the creation of variants by mutagenesis or transformationof sunflower plants producing seeds having low saturated fat content.

Aspects of the invention provide novel sunflower plants producing seedshaving low saturated fat content and high linoleic acid content. Thisinvention, in part, relates to the seeds of sunflower having lowsaturated fat content and high linoleic acid content, to the plants, orplant parts, of sunflower plants producing seeds having low saturatedfat content and high linoleic acid content, and to methods for producinga sunflower plant produced by crossing the sunflower plants producingseeds having low saturated fat content and high linoleic acid contentwith itself or another sunflower cultivar, and the creation of variantsby mutagenesis or transformation of sunflower plants producing seedshaving low saturated fat content and high linoleic acid content.

Examples of seeds having low saturated fat content include, but are notlimited to, seeds having about 2.8% or less, about 2.9% or less, about3% or less, about 3.1% or less, about 3.2% or less, or about 3.3% orless total combined palmitic acid (16:0) and stearic acid (18:0)content.

Examples of seeds of having low saturated fat content and high linoleicacid (18:2) content include, but are not limited to, seeds having about4.1% or less, about 5% or less, about 6% or less, about 7% or less,about 8% or less, about 9% or less, about 10% or less, about 11% orless, or about 12% or less total combined palmitic acids (16:0) andstearic acid (18:0) content and having about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, or about 74% or more linoleic acid (18:2).

Thus, any such methods using the sunflower plants that produce seedshaving low saturated fat and, optionally, high linoleic acid content,are part of this invention (e.g., selfing, backcrosses, hybridproduction, crosses to populations, and the like). All plants producedusing sunflower plants producing seeds having as a parent low saturatedfat and, optionally, high linoleic acid content, are within the scope ofthis invention. Advantageously, the sunflower plant could be used incrosses with other, different, sunflower plants to produce firstgeneration (F₁) sunflower hybrid seeds and plants with superiorcharacteristics.

In another aspect, the present invention provides for single or multiplegene converted sunflower plants producing seeds having low saturated fatand, optionally, high linoleic acid content. The transferred gene(s) maypreferably be a dominant or recessive allele. The transferred gene(s)can confer such traits as herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility, malesterility, enhanced nutritional quality, and industrial usage. The genemay be a naturally occurring sunflower gene or a transgene introducedthrough genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of sunflower plants producing seeds having lowsaturated fat and, optionally, high linoleic acid content. The tissueculture can be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing sunflower plantproducing seeds having low saturated fat and, optionally, high linoleicacid content, and of regenerating plants having substantially the samegenotype as the foregoing sunflower plant. The regenerable cells in suchtissue cultures can be embryos, protoplasts, meristematic cells, callus,pollen, leaves, anthers, roots, root tips, flowers, seeds, pods orstems. Still further, the present invention provides sunflower plantsregenerated from the tissue cultures of the invention.

In another aspect, the present invention provides a method ofintroducing a desired trait into sunflower plants producing seeds havinglow saturated fat and, optionally, high linoleic acid content, whereinthe method comprises: crossing a sunflower plant producing seeds havinglow saturated fat and, optionally, high linoleic acid content with aplant of another sunflower cultivar that comprises a desired trait toproduce F₁ progeny plants, wherein the desired trait is selected fromthe group consisting of male sterility, herbicide resistance, insectresistance, and resistance to bacterial disease, fungal disease or viraldisease; selecting one or more progeny plants that have the desiredtrait to produce selected progeny plants; crossing the selected progenyplants with the sunflower plants producing seeds having low saturatedfat and, optionally, high linoleic acid content to produce backcrossprogeny plants; selecting for backcross progeny plants that have thedesired trait and physiological and morphological characteristics ofsunflower plants producing seeds having low saturated fat and,optionally, high linoleic acid content to produce selected backcrossprogeny plants; and repeating these steps to produce selected first orhigher backcross progeny plants that comprise the desired trait and allof the physiological and morphological characteristics of sunflowerplants producing seeds having low saturated fat and, optionally, highlinoleic acid content.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fine mapping of the low stearic acid QTL in theHA1875-HA1865 interval of LG17 (Panel A: Maps of LG 17 with new markers(in blue color) and Panel B: Fine mapping of the low stearic acid QTL tothe HA1875-ORS565 interval);

FIG. 2 shows alignment of sequences of the KASII-2 gene from the twoparental lines showing SNPs and indels (IDs numbers 333.1 (SEQ ID NO:38)and 333.2 (SEQ ID NO:39) represented clones from OND163R amplicons, and332.4 (SEQ ID NO:40) and 332.5 (SEQ ID NO:41) from H280R[1]/687R-1-8-1amplicons);

FIG. 3 shows co-localization of the low palmitic acid QTL (Panel A) andfatty acid gene KASIII-2 (Panel B) on LG 5.

DETAILED DESCRIPTION OF THE INVENTION

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Elite sunflower. A sunflower cultivar which has been stabilized forcertain commercially important agronomic traits comprising a stabilizedyield of about 100% or greater relative to the yield of check varietiesin the same growing location growing at the same time and under the sameconditions. In one embodiment, “elite sunflower” means a sunflowercultivar stabilized for certain commercially important agronomic traitscomprising a stabilized yield of 110% or greater relative to the yieldof check varieties in the same growing location growing at the same timeand under the same conditions. In another embodiment, “elite sunflower”means a sunflower cultivar stabilized for certain commercially importantagronomic traits comprising a stabilized yield of 115% or greaterrelative to the yield of check varieties in the same growing locationgrowing at the same time and under the same conditions.

Embryo. The embryo is the small plant contained within a mature seed.

FAME analysis. Fatty Acid Methyl Ester analysis is a method that allowsfor accurate quantification of the fatty acids that make up complexlipid classes.

Imidazolinone resistance (Imi). Resistance and/or tolerance is conferredby one or more genes which alter acetolactate synthase (ALS), also knownas acetohydroxy acid synthase (AHAS) allowing the enzyme to resist theaction of imidazolinone.

Mutagenesis. Mutagenesis refers to mutagenesis of a plant or plant partwith a mutagen (e.g., a chemical or physical agent that increases thefrequency of mutations in a target plant or plant part). By way ofnon-limiting example, the double chemical mutagenesis technique ofKonzak, as described in U.S. Pat. No. 6,696,294 (the disclosure of whichis incorporated by reference herein), can be used to induce mutantalleles in endogenous plant genes.

Oil content. This is measured as percent of the whole dried seed and ischaracteristic of different varieties. It can be determined usingvarious analytical techniques such as NMR, NIR, and Soxhlet extraction.

Percentage of total fatty acids. This is determined by extracting asample of oil from seed, producing the methyl esters of fatty acidspresent in that oil sample and analyzing the proportions of the variousfatty acids in the sample using gas chromatography. The fatty acidcomposition can also be a distinguishing characteristic of a variety.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing, or via genetic engineering, wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single gene transferred intothe variety via the backcrossing technique or via genetic engineering.

Stabilized. Reproducibly passed from one generation to the nextgeneration of inbred plants of same variety.

Total Saturated (TOTSAT). Total percent oil of the seed of the saturatedfats in the oil including C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 andC24.0.

According to a particular embodiment the invention, there is provided anovel sunflower plant producing seeds having low saturated fat content.This embodiment relates to the seeds of sunflower having low saturatedfat content, to the plants, or plant parts, of sunflower plantsproducing seeds having low saturated fat content, and to methods forproducing a sunflower plant produced by crossing the sunflower plantproducing seeds having low saturated fat content with itself or anothersunflower cultivar, and the creation of variants by mutagenesis ortransformation of sunflower plants producing seeds having low saturatedfat content.

Other aspects of the invention provide novel sunflower plants producingseeds having low saturated fat content and high linoleic acid content.This embodiment relates to the seeds of sunflower having low saturatedfat content and high linoleic acid content, to the plants, or plantparts, of sunflower plants producing seeds having low saturated fatcontent and high linoleic acid content, and to methods for producing asunflower plant produced by crossing the sunflower plants producingseeds having low saturated fat content and high linoleic acid contentwith itself or another sunflower cultivar, and the creation of variantsby mutagenesis or transformation of sunflower plants producing seedshaving low saturated fat content and high linoleic acid content.

Examples of seeds having low saturated fat content include, but are notlimited to, seeds having about 2.8% or less, about 2.9% or less, about3% or less, about 3.1% or less, about 3.2% or less, or about 3.3% orless total combined palmitic acid (16:0) and stearic acid (18:0)content.

Examples of seeds of having low saturated fat content and high linoleicacid (18:2) content include, but are not limited to, seeds having about6% or less, about 4.1% or less, about 5% or less, about 6% or less,about 7% or less, about 8% or less, about 9% or less, about 10% or less,about 11% or less, or about 12% or less total combined palmitic acids(16:0) and stearic acid (18:0) content and having about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, or about 74% or more linoleic acid(18:2).

Thus, any such methods using the sunflower plants producing seeds havinglow saturated fat and, optionally, high linoleic acid content, are partof this invention (e.g., selfing, backcrosses, hybrid production,crosses to populations, and the like). All plants produced usingsunflower plants that produce seeds having as a parent low saturated fatand, optionally, high linoleic acid content, are within the scope ofthis invention. Advantageously, the sunflower plant could be used incrosses with other, different, sunflower plants to produce firstgeneration (F₁) sunflower hybrid seeds and plants with superiorcharacteristics.

In another aspect, the present invention provides for single or multiplegene converted sunflower plants producing seeds having low saturated fatand, optionally, high linoleic acid content. The transferred gene(s) maypreferably be a dominant or recessive allele. Preferably, thetransferred gene(s) will confer such traits as herbicide resistance,insect resistance, bacterial resistance, fungal resistance, viraldisease resistance, male fertility, male sterility, enhanced nutritionalquality, and industrial usage. The gene may be a naturally occurringsunflower gene or a transgene introduced through genetic engineeringtechniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of sunflower plants producing seeds having lowsaturated fat and, optionally, high linoleic acid content. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoingsunflower plant producing seeds having low saturated fat and,optionally, high linoleic acid content, and of regenerating plantshaving substantially the same genotype as the foregoing sunflower plant.The regenerable cells in such tissue cultures can be embryos,protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots,root tips, flowers, seeds, pods or stems. Still further, an embodimentof the invention provides sunflower plants regenerated from the tissuecultures of the invention.

In another aspect, the present invention provides a method ofintroducing a desired trait into sunflower plants producing seeds havinglow saturated fat and, optionally, high linoleic acid content, whereinthe method comprises: crossing a sunflower plant that produces seedshaving low saturated fat and, optionally, high linoleic acid contentwith a plant of another sunflower cultivar that comprises a desiredtrait to produce F₁ progeny plants, wherein the desired trait isselected from the group consisting of male sterility, herbicideresistance, insect resistance, and resistance to bacterial disease,fungal disease or viral disease; selecting one or more progeny plantsthat have the desired trait to produce selected progeny plants; crossingthe selected progeny plants with the sunflower plants producing seedshaving low saturated fat and, optionally, high linoleic acid content toproduce backcross progeny plants; selecting for backcross progeny plantsthat have the desired trait and physiological and morphologicalcharacteristics of sunflower plants that produce seeds having lowsaturated fat and, optionally, high linoleic acid content to produceelected backcross progeny plants; and repeating these steps to produceselected first or higher backcross progeny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of sunflower plants producing seeds having low saturatedfat and, optionally, high linoleic acid content.

Useful methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. Expression vectors can be introduced into plant tissues usingthe microprojectile media delivery with the biolistic deviceAgrobacterium-mediated transformation. Transformant plants obtained withthe protoplasm of the invention are intended to be within the scope ofthis invention.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional or modified versions of native orendogenous genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or cultivar.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNA thatincludes a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid and can be used alone or incombination with other plasmids to provide transformed sunflower plantsusing transformation methods as described below to incorporatetransgenes into the genetic material of the sunflower plant(s).

Expression Vectors for Sunflower Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection (i.e., inhibiting growth of cells that do not containthe selectable marker gene) or by positive selection (i.e., screeningfor the product encoded by the genetic marker). Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene under the control of plantregulatory signals, which confers resistance to kanamycin. See, e.g.,Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. See, e.g., Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferaseand the bleomycin resistance determinant. See Hayford et al., PlantPhysiol. 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol. 14:197 (1990); Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. See Comai etal., Nature 317:741-744 (1985); Gordon-Kamm et al., Plant Cell 2:603-618(1990); and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. See Eichholtz et al., Somatic Cell Mol. Genet.13:67 (1987); Shah et al., Science 233:478 (1986); Charest et al., PlantCell Rep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance, such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase. See,R. A. Jefferson, Plant Mol. Biol. Rep. 5:387 (1987); Teeri et al., EMBOJ. 8:343 (1989); Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987); DeBlock et al., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene, T. M. Green, p. 1-4(1993); and Nalewayet al., J. Cell Biol. 115:151a (1991). However, these in vivo methodsfor visualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. See, Chalfie et al., Science 263:802 (1994). GFP and mutants ofGFP may be used as screenable markers.

Expression Vectors for Sunflower Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNA that isupstream from the start of transcription and that is involved inrecognition and binding of RNA polymerase and other proteins to initiatetranscription. A “plant promoter” is a promoter capable of initiatingtranscription in plant cells. Examples of promoters under developmentalcontrol include promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, seeds, fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred.” Promoters which initiate transcription only incertain tissues are referred to as “tissue-specific.” A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression insunflower. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sunflower. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See, Wardet al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to: those from the ACEI systemthat responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2gene from maize that responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227:229-237 (1991); and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)); and Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression insunflower or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sunflower.

Different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto: the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)); the promoters from rice actingenes (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989), and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)); and maize H3 histone (Lepetit et al., Mol. Gen. Genetics231:276-285 (1992), and Atanassova et al., Plant Journal 2 (3):291-300(1992)). The ALS promoter, Xbal/NcoI fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to theXbal/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin sunflower. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in sunflower. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter can produce the protein product of the transgene exclusively,or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476-482 (1983), and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985), and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Transport of protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondrion or for secretion into the apoplast, can beaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment, or for secretion tothe apoplast. Many signal sequences are known in the art. See, e.g.,Becker et al., Plant Mol. Biol. 20:49 (1992); P. S. Close, Master'sThesis, Iowa State University (1993); C. Knox et al., “Structure andOrganization of Two Divergent Alpha-Amylase Genes from Barley,” PlantMol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., A short amino acid sequence able to specify nuclear location, Cell39:499-509 (1984); Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

In aspects of the invention, the transgenic plant provided forcommercial production of foreign protein is a sunflower plant. In otheraspects, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, agronomic genes can be expressed in transformed plants. Moreparticularly, plants can be genetically engineered to express variousphenotypes of agronomic interest. Exemplary genes that may be used inthis regard include, but are not limited to, those categorized below.

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A) Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance genes to engineer plants that are resistant to specificpathogen strains. See, e.g., Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B) A gene conferring resistance to a pest, such as soybean cystnematode. See, e.g., PCT Application WO 96/30517; PCT Application WO93/19181.

C) A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, e.g., Geiser et al., Gene 48:109(1986), which discloses the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D) A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

E) A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

F) An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, e.g., Abe et al., J. Biol. Chem. 262:16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor); Huubet al., Plant Molec. Biol. 21:985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I); Sumitani et al., Biosci.Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus .alpha.-amylase inhibitor); and U.S. Pat. No. 5,494,813(Hepher and Atkinson, issued Feb. 27, 1996).

G) An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., which discloses genes encoding insect-specific,paralytic neurotoxins.

I) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M) A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

N) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci. 89:43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

O) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

P) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R) A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide:

A) An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B) An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, such as:

A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B) Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize for example, this could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene); Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase); Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes); Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene); and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Sunflower Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Mild et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, B. R.Glick and J. E. Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, e.g., Gruber et al., “Vectors for Plant Transformation”in Methods in Plant Molecular Biology and Biotechnology, B. R. Glick andJ. E. Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

A) Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, e.g., Horsch et al., Science 227:1229(1985). A. tumefaciens and A. rhizogenes are plant pathogenic soilbacteria which genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,C. I. Kado, Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Mild et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B) Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987); J.C. Sanford, Trends Biotech. 6:299 (1988); Klein et al., Bio/Technology6:559-563 (1988); J. C. Sanford, Physiol. Plant 7:206 (1990); Klein etal., Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580(Christou, et al.), issued May 14, 1991; U.S. Pat. No. 5,322,783 (Tomes,et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J, 4:2731 (1985); Christou etal., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985), and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992), and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of sunflower target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety can then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular sunflower cultivar using theforegoing transformation techniques can be moved into another cultivarusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach can be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties which do not contain that gene. As used herein,“crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Tissue Culture of Sunflowers

Further production of a sunflower plant producing seeds having lowsaturated fat and, optionally, high linoleic acid content can occur byself-pollination or by tissue culture and regeneration. Tissue cultureof various tissues of sunflower and regeneration of plants therefrom isknown. For example, the propagation of a sunflower cultivar by tissueculture is described in U.S. Pat. No. 6,998,516.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to U.S. Pat. No. 6,998,516, which isincorporated herein in its entirety by reference. Thus, another aspectof this invention is to provide cells, which upon growth anddifferentiation, produce a sunflower plants having seeds containing lowsaturated fat and, optionally, high linoleic acid content.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type, or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures include protoplasts, calli, plant clumps, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as embryos, pollen, flowers, seeds, pods,leaves, stems, roots, root tips, anthers, and the like. Means forpreparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,2345,977,445, and 6,998,516 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

Single-Gene Converted (Conversion) Plants

When the term “sunflower plant” is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term “single gene converted plant” as used herein refers tothose sunflower plants which are developed by a plant breeding techniquecalled backcrossing, or via genetic engineering, wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single gene transferred intothe variety via the backcrossing technique. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the variety. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent(i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to therecurrent parent). The parental sunflower plant, which contributes thegene for the desired characteristic, is termed the “nonrecurrent” or“donor parent.” This terminology refers to the fact that thenonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental sunflower plant to which the geneor genes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a sunflower plant is obtained wherein essentially all of thedesired morphological and physiological characteristics of the recurrentparent are recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic and, therefore, the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross. One ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which arehereby incorporated by reference.

This invention also is directed to methods for producing a sunflowerplant by crossing a first parent sunflower plant with a second parentsunflower plant, wherein the first or second parent sunflower plant is asunflower plant producing seeds having low saturated fat and,optionally, high linoleic acid content. Further, both first and secondparent sunflower plants can originate from a sunflower plant producingseeds having low saturated fat and, optionally, high linoleic acidcontent. Thus, any such methods using a sunflower plant producing seedshaving low saturated fat and, optionally, high linoleic acid content arepart of this invention (i.e., selfing, backcrosses, hybrid production,crosses to populations, and the like). All plants produced using asunflower plant producing seeds having low saturated fat and,optionally, high linoleic acid content as a parent are within the scopeof this invention, including those developed from varieties derived froma sunflower plant producing seeds having low saturated fat and,optionally, high linoleic acid content. Advantageously, the sunflowervariety could be used in crosses with other, different, sunflower plantsto produce first generation (F₁) sunflower hybrid seeds and plants withsuperior characteristics. The variety of the invention can also be usedfor transformation where exogenous genes are introduced and expressed bythe variety of the invention. Genetic variants created either throughtraditional breeding methods using a sunflower plant producing seedshaving low saturated fat and, optionally, high linoleic acid content orthrough transformation of a sunflower plant producing seeds having lowsaturated fat and, optionally, high linoleic acid content by any of anumber of protocols known to those of skill in the art are intended tobe within the scope of this invention.

EXAMPLES

The present invention is further described in the following examples,which are offered by way of illustration and are not intended to limitthe invention in any manner.

Example 1 Sunflowers Producing Seeds Having Low Saturated Fat Content

Sunflower germplasm with unusually low saturate levels has beendeveloped through normal breeding techniques. Seed oil content ofsunflower cultivars are provided in Table 1.

TABLE 1 TOTAL C16:0 + Sample C16:0 C16:1 C18:0 C18:1 C18:2 SATS C18:0H757B/LS10670B-B-17-3-23.06 2.34 0.09 0.48 94.18 1.51 3.39 2.82H757B/LS10670B-B-17-3-33.11 2.47 0.11 0.51 93.62 2.11 3.42 2.98H757B/LS10670B-B-17-3-23.04 2.24 0.09 0.53 94.25 1.49 3.45 2.77H757B/LS10670B-B-17-3-02.08 2.70 0.13 0.50 93.26 2.24 3.67 3.2H757B/LS10670B-B-17-3-18.21 2.45 0.11 0.54 93.62 1.73 3.68 2.99HE06EE010716.001 2.17 0.11 0.82 94.29 1.41 3.63 2.99 HE06EE010834.0022.31 0.11 0.65 94.74 0.82 3.68 2.95 HE06EE010746.002 2.40 0.11 0.7293.87 1.03 3.68 3.12 HE06EE010700.003 2.48 0.13 0.57 93.46 1.78 3.783.05 HE06EE016032.005 2.42 0.10 0.64 92.86 1.82 3.82 3.06HE06EE016037.005 2.25 0.08 0.75 93.06 1.71 3.86 3.00 HE06EE016032.0022.40 0.10 0.70 93.00 1.72 3.87 3.09 HE06EE010717.002 2.44 0.10 0.8289.76 5.51 3.88 3.26 HE06EE010695.001 2.48 0.12 0.66 91.93 3.20 3.883.14 HE06EE010816.002 2.34 0.12 0.88 94.10 1.24 3.88 3.22HE06EE010700.001 2.48 0.14 0.65 94.31 0.89 3.90 3.13 HE06EE010814.0022.46 0.10 0.79 94.11 1.19 3.91 3.24 HE06EE010760.004 2.54 0.11 0.6394.07 1.16 3.92 3.16 HE06EE010741.003 2.34 0.11 0.93 94.51 0.73 3.933.26 HE06EE010737.003 2.33 0.13 0.96 93.53 1.12 3.93 3.29HE06EE016050.005 2.41 0.08 0.73 92.57 2.67 3.94 3.13 HE06EE016032.0042.44 0.11 0.63 92.49 1.80 3.94 3.07 HE06EE010763.002 2.43 0.11 0.7894.28 0.98 3.94 3.21 HE06EE010829.002 2.53 0.13 0.70 93.26 1.84 3.953.23 HE06EE010738.002 2.78 0.15 0.62 89.75 5.22 3.96 3.40HE06EE010741.004 2.42 0.11 0.88 94.10 0.61 3.96 3.30 HE06EE010824.0042.35 0.10 0.80 94.14 1.15 3.97 3.15 HE06EE010745.003 2.81 0.11 0.6888.66 6.32 3.98 3.48 HE06EE010816.001 2.52 0.11 0.80 91.45 3.77 3.983.32

Example 2 Sunflowers Producing Seeds Having Low Saturated Fat Contentand High Linoleic Acid Content

Sunflower germplasm with unusually low saturate levels has beendeveloped through normal breeding techniques. Seed oil content ofsunflower cultivars are provided in Table 2.

TABLE 2 TOTAL C16:0 + Sample C16:0 C16:1 C18:0 C18:1 C18:2 SATS C18:0H757B/LS10670B-B-17-3-14.01 4.25 0.09 1.13 37.87 55.45 5.90 5.38H757B/LS10670B-B-17-3-02.18 4.80 0.11 0.68 39.63 53.55 6.05 5.48H757B/LS10670B-B-17-3-27.12 4.01 0.08 1.37 38.48 54.68 6.07 5.38H757B/LS10670B-B-17-3-16.02 5.19 0.14 0.73 35.14 57.79 6.22 5.92H757B/LS10670B-B-17-3-36.22 4.99 0.09 1.25 17.97 74.37 6.81 6.24

Example 3 Sunflowers Producing Seeds Having Low Saturated Fat Content

Sunflower germplasm with unusually low saturate levels has beendeveloped through normal breeding techniques. Seed oil content ofsunflower cultivars are provided in Table 3.

TABLE 3 TOTAL Sample C16:0 C16:1 C18:0 C18:1 C18:2 SATS NuSun/NoSaturate NS1982.16/OND163R-1-05 2.29 0.05 0.65 67.37 28.19 3.48 NS1982.82.09 0.08 0.55 79.40 15.99 3.10 No Saturate/High Oleic NS1982.8-03 1.600.03 0.37 95.13 1.48 2.33 NS1982.8 1.63 0.07 0.41 94.81 1.26 2.48H117R[4]//H757B/LS10670B/// 1.79 0.05 0.29 95.30 0.84 2.57NS1982.6-2-023-1-12-076 Low Saturate/Linoleic CND117R/NS1982.8-3-06 5.290.07 0.73 18.19 74.43 6.41 OI1601B[2]//H757B/LS10670B[1]/// 3.76 0.070.80 34.97 58.62 5.29 NS1982.6 = B-3-04 CN2343B/4/CN2343B[2]// 3.13 0.022.07 36.03 56.65 6.23 H757B/LS10670B///NS1982.11#1#1-3-11 Low StearicNS1982.8/OND163R-2-12-009 2.75 0.66 0.25 92.95 1.99 3.43H117R[4]//H757B/LS10670B/// 1.90 0.04 0.27 95.03 1.00 2.65NS1982.6-2-023-1-12-038 OID263R/NS1982.8-4-12-002 3.08 0.12 0.27 93.541.48 3.87 Low Palmitic H251B[2]/IAST-4 = 1 = 100// 1.47 0.24 2.59 92.590.65 5.42 NS1982.16-11-39-041 NS1982.14-08 1.51 0.02 2.24 92.84 1.354.90 NS1982.16 1.52 0.06 1.05 94.37 0.85 3.39 Very High OleicH117R[4]//H757B/LS10670B// 1.79 0.05 0.29 95.30 0.84 2.57NS1982.6-2-023-1-12-076 NS1982.8/OND163R-2-12-059 1.87 0.10 0.44 95.220.97 2.76 ON3351B/NS1982.8-1-04 2.04 0.03 0.50 95.20 0.70 3.08

As can be seen in Table 3, the data demonstrates seed oil having totalsaturates as low as 2.33% in a high oleic (>80%) background, no Saturate(<3.5%) profile in a NuSun (55-50% oleic) background, oleic levels up to95.30%; stearic levels as low as 0.25%, palmitic levels as low as 1.47%,and low Saturate (<7.0%) profile in a linoleic (<55% oleic) background.

Example 4 Sunflowers Producing Seeds Having Low Saturated Fat, StearicAcid, and Palmitic Acid Content

Sunflower germplasm with unusually low saturate levels has beendeveloped through normal breeding techniques. Seed oil content ofsunflower cultivars are provided in Table 4.

TABLE 4 Name C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1NS1982.8/OND163R-12-90 1.37 0.01 1.70 91.93 2.83 0.08 0.21 0.60H117R[4]//H757B/LS10670B-B-17-3-23 = 1.39 0.02 0.53 94.89 1.55 0.08 0.090.66 B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/LS10670B-B-17-3-23= 1.44 0.03 0.36 94.83 1.84 0.09 0.08 0.74 B1 = 2 =16///NS1982.6-2-23.1-1 H117R[4]//H757B/LS10670B-B-17-3-23 = 1.58 0.020.24 94.54 2.05 0.10 0.06 0.79 B1 = 2 = 16///NS1982.6-2-23.1-1H117R[4]//H757B/LS10670B-B-17-3-23 = 1.89 0.03 0.24 94.17 2.31 0.13 0.040.70 B1 = 2 = 16///NS1982.6-2-23.1-1 H117R[4]//H757B/LS10670B-B-17-3-23= 1.94 0.03 0.23 94.58 1.80 0.12 0.07 0.69 B1 = 2 =16///NS1982.6-2-23.1-1 Name C20:2 C22:0 C22:1 C24:0 C24:1 TSatsNS1982.8/OND163R-12-90 nd 0.70 0.01 0.33 nd 4.32H117R[4]//H757B/LS10670B-B-17-3-23 = nd 0.40 0.03 0.19 nd 2.60 B1 = 2 =16///NS1982.6-2-23.1-1 H117R[4]//H757B/LS10670B-B-17-3-23 = nd 0.31 0.020.14 nd 2.33 B1 = 2 = 16///NS1982.6-2-23.1-1H117R[4]//H757B/LS10670B-B-17-3-23 = nd 0.24 0.04 0.15 nd 2.28 B1 = 2 =16///NS1982.6-2-23.1-1 H117R[4]//H757B/LS10670B-B-17-3-23 = nd 0.21 0.030.11 nd 2.50 B1 = 2 = 16///NS1982.6-2-23.1-1H117R[4]//H757B/LS10670B-B-17-3-23 = nd 0.22 0.03 0.13 nd 2.60 B1 = 2 =16///NS1982.6-2-23.1-1

As can be seen in Table 4, this set of data includes the low values forstearic (0.23%), palmitic (1.37%), and total saturated oils (2.28%).

Example 5 Marker Development for Low Stearic and Low Palmitic

A strategy for marker development was developed as described herein.First, markers from the target QTL regions, developed at DowAgroSciences as well as from the public resources, were identified andscreened for polymorphisms between the parental lines of correspondingmapping populations. Polymorphic markers were then screened in themapping populations. For monomorphic (non-informative) markers, primerswere designed to amplify their corresponding genomic loci and theamplicons were sequenced to identify single nucleotide polymorphisms(SNPs), if any, between the parental lines. TaqMan MGB AllelicDiscrimination assays were developed for the identified SNPs and weremapped on the respective population. Second, based on sequences ofcandidate genes for fatty acids, primers flanking introns were designedto isolate fatty acid gene sequences from the parental lines. Nucleotidepolymorphisms at the sequence level were developed into markers based ontheir polymorphic nature and were then screened in the mappingpopulations. JoinMap 3.0 (Van Ooijen, 2004a) was employed to map thenewly developed markers, and MapQTL 5 (Van Ooijen, 2004b) was used tofine map QTLs.

A) Marker Development for Low Stearic Acid

SSR marker development: Eight SSR markers were screened forpolymorphisms between parental lines ONN687R andH757B/LS10760B-B-17-3-23-5 of the ONN687R×H757B/LS10760B-B-17-3-23-5mapping population which was previously used to map the target lowstearic acid QTL. (See Table 5.) Four SSR markers, HA0442, CRT22, ORS565and ORS732, were polymorphic. HA0442 and CRT22 amplicons from ONN687Rand H757B/LS10760B-B-17-3-23-5 were resolved on ABI 3730 sequencer, andwere 163 bp and 165 bp, respectively, for marker HA0442, and 290 bp and261 bp, respectively, for CRT22. ORS565 and ORS732 amplicons fromONN687R and H757B/LS10760B-B-17-3-23-5 were resolved on 3% Metaphorgels. The corresponding mapping populationONN687R×H757B/LS10760B-B-17-3-23-5 was genotyped with HA0442, CRT22,ORS565 and ORS732 using the following PCR primers and reactionconditions.

HA0442 Forward Primer: (SEQ ID NO: 1) 5′-HEX-TGGAACTGTAAATGGACCCAAG-3′HA0442 Reverse Primer: (SEQ ID NO: 2) 5′-GCACTGCACCATTTATGAGAAG-3′CRT22 Forward Primer: (SEQ ID NO: 3) 5′-HEX-TCGAGATGAAACCGAATGAAGAAA-3′CRT22 Reverse Primer: (SEQ ID NO: 4) 5′-GTTTCTTGGGACTGATATTGCCAAGTGGG-3′ORS565 Forward Primer: (SEQ ID NO: 5) 5′-TGGTCAACGGATTTAGAGTCAA-3′ORS565 Reverse Primer: (SEQ ID NO: 6) 5′-TCCAGTTTGGTCTTGATTTGG-3′ORS732 Forward Primer: (SEQ ID NO: 7) 5′-GCACGGAACTCCTCAAATGT-3′ORS732 Reverse Primer: (SEQ ID NO: 8) 5′-GCACGGGAAACAAAGAGTCA-3′

PCR Components:

-   -   4 ng gDNA    -   1×PCR buffer (Qiagen, Valencia, Calif.)    -   0.25 μM Forward primer    -   0.25 μM Reverse primer    -   1 mM MgCl₂    -   0.1 mM of each dNTP    -   0.4% PVP    -   0.04 Units HotStar Taq DNA polymerase (Qiagen, Valencia, Calif.)    -   Total Volume: 4.8 μl

Thermocycler Setup:

-   -   Step 1: 94° C. for 12 minutes    -   Step 2: 94° C. for 30 seconds    -   Step 3: 55° C. for 30 seconds    -   Step 4: 72° C. for 30 seconds    -   Step 5: repeat steps 2, 3 and 4 for 35 cycles    -   Step 6: 72° C. for 30 minutes

SNP marker development: Eight pairs of primers were used to amplifyeight genomic loci from both ONN687R and H757B/LS10760B-B-17-3-23-5 todevelop SNP markers (Table 6). Three primer pairs (ZVG76snpF/R,ZVG77snpF/R, and ZVG78snpF/R) were designed based on sequences fromrestriction fragment length polymorphism (RFLP) probes ZVG76, ZVG77 andZVG78 (Kolkman et al. 2007). Primer sequences for HT57F/R, HT64F/R,HT131F/R, HT134F/R, and HT210F/R were from Lai et al. (2005). SNPs werefound in the amplicons from HT64F/R, HT210F/R, and ZVG78snpF/R. TaqManMGB Allelic Discrimination assays were developed for one SNP locus inthe HT64F/R amplicon and one SNP locus in the ZVG78snpF/R amplicon (Seebelow), and the ONN687R×H757B/LS10760B-B-17-3-23-5 mapping populationwas genotyped with those two SNP markers using the developed TaqManassays.

There were four SNP loci (marked in bold) in the HT64F/R amplicons fromONN687R and H757B/LS10760B-B-17-3-23-5. The TaqMan Assay was developedfor the R-locus. The sequences for Forward Primer, Reverse Primer, Probe1 and Probe 2 are 5′-CCGGCTGCTTCTAGACCTTATAAG-3′ (SEQ ID NO:9),5′-TCGTCGGTGGGACACACA-3′ (SEQ ID NO:10), 5′-6FAM-ACTGTTGGATCGGTTC-3′(SEQ ID NO:11), and 5′-VIC-CACTGTTGGATCGATT-3′ (SEQ ID NO:12),respectively.

(SEQ ID NO: 13) TTATTCTCGGCTTCCGGTGTGATTTTACTCTCATGGTTAAGTTTTCAAGAGATTGTCGCY(T/C)GCTGAAAACTTTTTATATTGTTTCGGTATGATCTTGGAGTTTATAGCCTTTGTAAGGTTAAGAATGAAACACCCGGCTGCTTCTAGACCTTATAAGATACCCGTGGGCACTGTTGGATCGR(A/G)TTCTTCTGTGTGTCCCACCGACGATTTTGATCTGTGTCGTGTTGGCTCTTTCTTCACTCAAGGTCATGATCGTTAGY(T/C)GTY(C/T)ATTGCCATATTTTTCGGGTTCGCATTGCAACCGTTTTTAAAGTTTGCCGAGAAGAAAAGATG GCTTAAATTTTCAACTAAAGCCGATCTTCCCG

There were also four SNP loci (marked in bold) in the ZVG78snpF/Ramplicons from ONN687R and H757B/LS10760B-B-17-3-23-5. The TaqMan Assaywas developed for the R-locus at the 5′ end. The sequences for ForwardPrimer, Reverse Primer, Probe 1 and Probe 2 are5′-GTCCATCTTTCCTCAACGACTTG-3′ (SEQ ID NO:14),5′-CCTAAACGCCTCGAAAAAGCT-3′ (SEQ ID NO:15),5′-6FAM-TTACCATGTCTATAATGC-3′ (SEQ ID NO:16), and5′-VIC-ATTACCATGTCTGTAATGC-3′ (SEQ ID NO:17), respectively.

(SEQ ID NO: 18) AACTGAGTTCTGTACGCCAGAGATTTGCCCGACCATGACCGCAGGTCCAAAGTAAGTCTTGCTATTGCACATTTGCACGATTAACGGTTTCTTATATAGAAGATACATGATTCTTGAATTTATGTAAATAAAACTTGACAGATATGAATACCGATGGGCTGATGGTGTGCAAATCAAGAAGCCTATTGAAGTTTCGGCTCCAAAGTACGTAGAGTTCTTGATGGATTGGATTGAGTCACAATTGGATGACGAGTCCATCTTTCCTCAACGACTTGGTAATTAGTTAATTACCATGTCTR(G/A)TAATGCATCATTTAATAAAGCTTTTTCGAGGCGTTTAGGAAACTGAAATAGTAATTTTCGATTGY(T/C)CGTGCAGGAGCGCCATTTCCCGCCAATTTTAGGGACGTTGTGAAAACGATATTTAAACGCTTGTTTCGTGTATAY(T/C)GCGCATATCTACCACACR(G/A)CATTTTCAGAAGATTGTGAGTCTTAAAGAAGAAGCCCATCTAAACACTTGTTTCAAGCATTT  CATATTGTTTACATGTGTAA

The following PCR setup was used for both SNP markers.

Real-Time PCR Components:

-   -   25 ng gDNA    -   1× Taqman Universal PCR Master Mix    -   22.5 μM Forward Primer    -   22.5 μM Reverse Primer    -   5 μM Probe 1    -   5 μM Probe 2    -   Total Volume: 25 μl

Bio-Rad iCycler Setup:

-   -   Step 1: 95° C. for 15 minutes    -   Step 2: 94° C. for 30 seconds    -   Step 3: 60° C. for 1 minute    -   Step 4: repeat steps 2 and 3 for 65 cycles    -   Step 5: 4° C. forever

Indel marker development: Primers were designed to amplify and sequence32 fatty acid related genes from the two parental lines ONN687R andH757B/LS10760B-B-17-3-23-5. Seven genes had polymorphisms, four geneshad weak amplifications, and all others were monomorphic (Table 6). Themapping population ONN687R×H757B/LS10760B-B-17-3-23-5 was screened withall identified polymorphisms.

Mapping new markers and fine mapping low stearic acid QTL: JoinMap 3.0(Van Ooijen, 2004a) was used to map all newly identified polymorphicmarkers. Marker CRT22 gave a significant segregation distortion and wasnot mapped. Six markers developed from the candidate gene approachmapped to chromosomes other than the target chromosome 17 (Table 6).Seven markers HA0442, ORS565, HT64, ZVG78, KASI-2, KASI-4, and ORS732were mapped to chromosome 17. Fatty acid genes KASI-2 and KASI-4 weremapped to chromosome 17 but not close to the target low stearic acid QTL(FIG. 1). With the newly mapped markers, the low stearic QTL was finemapped with MapQTL 5 ((Van Ooijen, 2004b) in the HA1875-ORS565 intervalwhich spanned 27 cM in the upper telomeric region of LG 17. The finemapped QTL had a significant LOD score of 23.2 and explained 50.8% ofthe variation in stearic acid content. The newly mapped markers can beused to facilitate the selection for low stearic acid in breedingprogram.

B) Developing and Mapping an Indel Marker for Palmitic Acid QTL

SNPs and indels were observed in the amplicon sequences of parentallines H280R[1]/687R-1-8-1 and OND163R with the primer pair for fattyacid gene KASIII-2 (Table 6, FIG. 2). The mapping populationH280R[1]/687R-1-8-1×OND163R was screened with this primer pair andamplicons were resolved on 3% Metaphor gels. Mapping program JoinMap 3.0(Van Ooijen, 2004a) located this indel marker inside the low palmiticacid QTL on linkage group 5 (FIG. 3).

TABLE 5List of markers investigated to saturate the low stearic acid QTL region.F Name Sequence R Name Sequence Note 1) SSR HA0953F-CAAACCAACAACCACCATCA  HA0953R AAACGACACCGATGAGAACC  Monomorphic HEX(SEQ ID NO: 34) (SEQ ID NO: 35) HA1909F- CTGAGTTTCGTGTACCATTTCTATTG HA1909R ACACCAATCAGTGGGTTTCATC   Poor marker FAM (SEQ ID NO: 36)(SEQ ID NO: 37) HA0442F- TGGAACTGTAAATGGACCCAAG  HA0442GCACTGCACCATTTATGAGAAG  Polymorphic HEX (SEQ ID NO: 1) (SEQ ID NO: 2)CRT22F- TCGAGATGAAACCGAATGAAGAAA  CRT22R GTTTCTTGGGACTGATATTGCCAAGTGGGPolymorphic HEX (SEQ ID NO: 3) (SEQ ID NO: 4) ORS297F-TGCAAAGCTCACACTAACCTG ORS297R GTGTCTGCACGAACTGTGGT Monomorphic FAMZVG76ssrF-  GCACCCTAGAGCTTCATTCG ZVG76ssrR AGCCCAAGGATGTTGTTTTGMonomorphic FAM ORS565F TGGTCAACGGATTTAGAGTCAA  ORS565RTCCAGTTTGGTCTTGATTTGG  Polymorphic (SEQ ID NO: 5) (SEQ ID NO: 6) ORS732FGCACGGAACTCCTCAAATGT  ORS732R GCACGGGAAACAAAGAGTCA  Polymorphic(SEQ ID NO: 7) (SEQ ID NO: 8) 2) SNP HT57F GCGATTATTGTTATGGACGC  HT57RAGCGGAAACTGTTCTTGTTG  Monomorphic (SEQ ID NO: 19) (SEQ ID NO: 20) HT64FTTATTCTCGGCTTCCGGT  HT64R CGGGAAGATCGGCTTTAG  SNPs (SEQ ID NO: 21)(SEQ ID NO: 22) HT131F CGTAACATGCAAGTTGTGGA  HT131RTGTACTCTAAACGGGCAACC  Monomorphic (SEQ ID NO: 23) (SEQ ID NO: 24) HT134FAGTCATGCTTGAAGGAGCTG  HT134R CTCTGTCAGCTTGCAATGAA  Monomorphic(SEQ ID NO: 25) (SEQ ID NO: 26) HT210F CTAAAACTGTCGCAAGGGAA  HT210RCCTCCATCAATGGTAAGCAC  SNPs (SEQ ID NO: 27) (SEQ ID NO: 28) ZVG76snpFTCCAACTCATGAACGGACTCT  ZVG76snpR Same as ZVG76ssrR Monomorphic(SEQ ID NO: 29) ZVG77snpF TTGGTGACTCTTGCAGCATC  ZVG77snpRAAGTTTAAAACCGCGTCGTG  Monomorphic (SEQ ID NO: 30) (SEQ ID NO: 31)ZVG78snpF TATGAGCCTCTTCGGTCTCG  ZVG78snpR CACCTTATTCAGCCCCGATA  SNPS(SEQ ID NO: 32) (SEQ ID NO: 33)

TABLE 6 Fatty acids genes investigated. Marker Enzyme Results on stearicpopulation Map position KASIII-1 Ketoacyl-ACP Syntetase III Co-dominantpolymorphism LG5 KASIII-2 Ketoacyl-ACP Syntetase III Non-polymorphicKASIII-3 Ketoacyl-ACP Syntetase III Weak amplification KASI-1Ketoacyl-ACP Syntetase I Non-polymorphic KASI-2 Ketoacyl-ACP Syntetase ICo-dominant polymorphism LG17 KASI-4 Ketoacyl-ACP Syntetase ICo-dominant polymorphism LG17 KASI-3 Ketoacyl-ACP Syntetase I Weakamplification KASII-1 Ketoacyl-ACP Syntetase II Non-polymorphic KASII-2Ketoacyl-ACP Syntetase II Weak amplification KASII-3 Ketoacyl-ACPSyntetase II Co-dominant polymorphism LG9 KAR Ketoacyl reductaseNon-polymorphic HAD Hyroxyacyl-ACP dehydratase Non-polymorphic Ear1Enoyl-ACP reductase Non-polymorphic FATA-1 FATA thioesteraseNon-polymorphic FATA-2 FATA thioesterase Non-polymorphic FATA-3 FATAthioesterase Dominant polymorphism LG7 FATB-1 FATB thioesterase Weakamplification FATB-2 FATB thioesterase Non-polymorphic CT-alpha1ACC−>carboxyltransferase-alpha (accA) Dominant polymorphism LG10 BCCPACC−>biotin carboxyl carrier protein (aacB) Non-polymorphic KCS1Ketoacyl-CoA synthase-I Non-polymorphic KCS2 Ketoacyl-CoA synthase-IINon-polymorphic KCS3 Ketoacyl-CoA synthase-III Non-polymorphic SAD 17Stearoyl-ACP desaturase Co-dominant polymorphism LG1 erLPATLysophosphatidic acid acyl transferase Non-polymorphic erPAP Phophatidicacid acyl transferase Non-polymorphic PDPSPhosphatidylglycerophosphatase synthase Non-polymorphic erLDS ERlinoleate desaturase Non-polymorphic FAD6-1 Plastid oleate desaturaseNon-polymorphic FAD6-2 Plastid oleate desaturase Non-polymorphicFAD2-1F5-R2 Oleate desaturase Non-polymorphic FAD2-1F5-R3 Oleatedesaturase Non-polymorphic

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims.

What is claimed is:
 1. A seed of a sunflower cultivar, wherein the oilcontent of the seed comprises 10% or less total combined palmitic acidand stearic acid content and about 15% or more linoleic acid content,wherein the genome of the sunflower cultivar seed comprises thepolynucleotide of SEQ ID NO: 13, and wherein the nucleotide at position60 of the polynucleotide is a cytosine (C), the nucleotide at position181 of the polynucleotide is a guanine (G), the nucleotide at position258 of the polynucleotide is a cytosine (C), and the nucleotide atposition 261 of the polynucleotide is a thymine (T).
 2. The seed of asunflower cultivar according to claim 1, wherein the total combinedpalmitic acid and stearic acid content is less than 10%, and wherein thetotal linoleic acid content is more than about 20%.
 3. The seed of asunflower cultivar according to claim 1, wherein the total combinedpalmitic acid and stearic acid content is less than about 4.1%, andwherein the total linoleic acid content is more than about 74%.
 4. Asunflower plant, or a part thereof, produced by growing the seed ofclaim 1, wherein said sunflower plant or a part thereof comprises saidSEQ ID NO: 13, and having seed with said oil content.
 5. A sunflowerinbred plant stabilized for seed oil comprising 12% or less totalcombined palmitic acid and stearic acid content and about 15% or morelinoleic acid content, wherein the genome of the sunflower inbred plantand seed obtained thereof comprises the polynucleotide of SEQ ID NO: 18,and wherein the nucleotide at position 299 of the polynucleotide is anadenine (A), the nucleotide at position 364 of the polynucleotide is acytosine (C), the nucleotide at position 439 of the polynucleotide is acytosine (C), and the nucleotide at position 457 of the polynucleotideis an adenine (A).
 6. The sunflower inbred plant stabilized for seed oilaccording to claim 5, wherein the seed oil comprises a total combinedpalmitic acid and stearic acid content of 11% or less, and a totallinoleic acid content of about or more than 20%.
 7. An inbred sunflowerseed of the sunflower inbred plant of claim 5, wherein the oil contentof the seed comprises 12% or less total combined palmitic acid andstearic acid content and about 15% or more linoleic acid content.
 8. Theinbred sunflower seed of claim 7, wherein the total combined palmiticacid and stearic acid content is less than 11%, and wherein the totallinoleic acid content is more than about 20%.
 9. A method for producinga hybrid sunflower seed, the method comprising crossing the sunflowerinbred plant of claim 5 with another, different sunflower plant, andobtaining the hybrid sunflower seed, and wherein said hybrid sunflowerseed comprises said SEQ ID NO: 18 in its genome and further comprisessaid stabilized seed oil.
 10. A hybrid sunflower seed produced by themethod according to claim 9, and wherein said hybrid sunflower seedcomprises said SEQ ID NO: 18 in its genome and further comprises saidstabilized seed oil.